Cabin1 domain-containing gene picd-1 interacts with pry-1/Axin to regulate multiple processes in Caenorhabditis elegans

The Axin family of scaffolding proteins control diverse processes, such as facilitating the interactions between cellular components and providing specificity to signaling pathways. While several Axin family members have been discovered in metazoans and shown to play crucial roles, their mechanism of action are not well understood. The Caenorhabditis elegans Axin homolog, pry-1, is a powerful tool for identifying interacting genes and downstream effectors that function in a conserved manner to regulate Axin-mediated signaling. Our lab and others have established pry-1’s essential role in developmental processes that affect the reproductive system, seam cells, and a posterior P lineage cell, P11.p. Additionally, pry-1 is crucial for lipid metabolism, stress responses, and aging. In this study, we expanded on our previous work on pry-1 by reporting a novel interacting gene named picd-1 (pry-1-interacting and Cabin1 domain-containing). PICD-1 protein shares sequence conservation with CABIN1, a component of the HUCA complex. Our findings have revealed that PICD-1 is involved in several pry-1-mediated processes, including stress response and lifespan maintenance. picd-1’s expression overlapped with that of pry-1 in multiple tissues throughout the lifespan. Furthermore, PRY-1 and PICD-1 inhibited CREB-regulated transcriptional coactivator homolog CRTC-1, which promotes longevity in a calcineurin-dependent manner. Overall, our study has demonstrated that picd-1 is necessary for mediating pry-1 function and provides the basis to investigate whether Cabin-1 domain-containing protein plays a similar role in Axin signaling in other systems.

Signaling pathways enable cells to communicate with each other and their environment. Because of their essential role in cells, these pathway components are tightly regulated via interaction with a host of cellular factors. The Axin family is a group of scaffolding proteins that bring together different proteins to facilitate their interactions and regulate their activity 1 . There are two Axin homologs in mammals: Axin1 and Axin2. Axin was initially discovered as a negative regulator of the WNT-mediated signaling cascade. However, subsequent studies revealed a much broader role of these proteins in other pathways, including JNK, AMPK, and TGFβ [1][2][3][4] . Axin regulates several processes, including organogenesis, anterior-posterior axis formation, neuronal development, and metabolic homeostasis. Further, loss of Axin function causes lethality, neuroectodermal defects, abnormal body axis patterning, and reduced adipogenesis 1 . However, the mechanism by which Axin regulates different biological processes and mediates specific interactions is not well understood.
In the nematode C. elegans, the Axin homolog PRY-1 controls processes such as embryogenesis, neuronal differentiation, vulval development, P11.p cell fate, and seam cell development 1,[5][6][7] . The WNT-dependent function of PRY-1 in vulval cells involves its interactions with several other proteins to form a destruction complex that results in the phosphorylation and degradation of BAR-1 (β-Catenin) 5 . However, little is known about the factors that interact with PRY-1 in WNT-independent processes. A comprehensive understanding of pry-1 function will require identification of its interacting proteins and downstream effectors. To this end, we previously performed a transcriptome profiling of pry-1 that revealed novel interacting partners and genetic network of pry-1 that regulate post-developmental events 6,8 . Specifically, we showed that PRY-1 is crucial for lipid metabolism, stress response, and lifespan maintenance, where it interacts with WNT-independent signaling pathway components 4,[8][9][10][11] . These include SBP-1/SREBP and vitellogenin involved in fatty acid synthesis and lipid storage, AAK-2/AMPK in the muscle that non-autonomously activates DAF-16/FOXO in the intestine and delays aging 8,10 , and the components of the endoplasmic reticulum unfolded protein response (ER-UPR) pathway.

Mutations in picd-1 do not affect vulval induction but cause morphogenetic defects.
In addition to using the gk3701 strain to examine mutant phenotypes, we generated a new allele, bh40, which has multiple in-frame stop codons in exon 1 (see "Methods" and Fig. 2A,B). qPCR analysis showed that bh40 and gk3701 greatly reduced picd-1 transcript levels (Fig. 2C). Interestingly, while the Pvl phenotype of pry-1(mu38) was enhanced by both the alleles (Fig. 1A,B), neither had an obvious impact on the penetrance of the multivulva (Muv) phenotype in pry-1(mu38) animals. In fact, the double mutants showed a slightly less Muv phenotype compared to pry-1(mu38) alone (Table 1, Fig. 1B), which may be due to changes in morphogenetic processes as vulval precursor induction is not affected by any of the picd-1 mutations (Fig. 2D, Table 1). Similar phenotypes were observed following picd-1 RNAi ( Figure S2). In agreement with this, picd-1(bh40), but not picd-1(gk3701), exhibited abnormal vulval invagination (Fig. 3A) and the vulval morphology phenotype of pry-1(mu38); picd- Table 1. Analysis of VPC induction, P12.pa cell fate, Pvl, and Muv penetrance in different strains. Each data consists of two independent replicates. Statistical analyses were done using one way-ANOVA. p values are indicated in brackets. n.s., not significant. While pry-1 and picd-1 mutants were compared to N2 control, pry-1; picd-1 double mutants were compared to respective pry-1 alleles. *Extra P12.pa cell in the place of P11.p. N number of animals examined. www.nature.com/scientificreports/ 1(bh40) double mutant was more severe compared to pry-1(mu38) alone. Furthermore, adult picd-1 mutants were weakly Pvl ( Fig. 3B and Table 1). Phenotypic analysis of both picd-1 mutant strains did not reveal any obvious defects in movement, feeding, and other characteristics. However, careful examination showed that this gene is involved in the development  Table 1). (C) Line graph showing the percentage of control and picd-1(bh40) animals with Egl phenotype at 20 °C and 25 °C. (B, C) Data represent a cumulative of two replicates (n = 64 for picd-1(bh40) and 58 for N2) and error bars represent the standard deviation. Statistical analyses were done using an unpaired t-test and significant differences are indicated by stars (*): * (p < 0.05), ** (p < 0.01). www.nature.com/scientificreports/ of the egg-laying system. The picd-1(bh40) worms were weakly egg-laying defective (Egl) (Fig. 3C, Video S2), and their Egl and Pvl phenotypes were enhanced at 25 °C (Fig. 3B,C). No Egl phenotype was observed in the picd-1(gk3701) strain. While more experiments are needed to determine the molecular basis of phenotypic differences between the two alleles, the data collectively demonstrate that picd-1 is required for the development of the reproductive system.

picd-1 is expressed in multiple tissues.
To gain further insights into the function of picd-1, we created a stable line carrying a picd-1::GFP transcriptional reporter. The analysis of transgenic animals revealed GFP fluorescence in developing tissues and organs, such as the pharynx, intestine, body wall muscles, hypodermis (seam cells), gonads, and vulva (Fig. 4A). This expression pattern resembled that of pry-1, which was recently described by our group 10 . As picd-1::GFP animals entered adulthood, fluorescence was localized to the intestine and certain head neurons (Fig. 4A), which persisted throughout the life of the animals. A broad range of picd-1 expression was also supported by previously published RNA-sequencing and microarray studies 19,20 . Overall, our expression analysis suggests that picd-1 functions in multiple tissues and may play a role in pry-1-mediated developmental and post -developmental processes.
To further elucidate the stress sensitivity of animals lacking picd-1 function, we examined the survivability of the animals following chemical treatments. Both gk3701 and bh40 alleles were sensitive to paraquat and tunicamycin, although the effect was more pronounced following paraquat exposure (Fig. 8D,E). We also tested paraquat sensitivity of pry-1(mu38); picd-1(bh40) double mutants and found no difference compared to pry-1(mu38) alone (Fig. 8F), which could be explained by the significantly reduced expression of picd-1 in pry-1 mutants.
As increased stress sensitivity can affect the lifespan of an animal, and pry-1 mutants are short-lived, we analyzed whether picd-1 plays a role in aging. Neither picd-1(gk3701) nor picd-1(RNAi) enhanced the lifespan defects of animals lacking pry-1 function (Fig. 9A,B, Table 2). Considering that pry-1 mutant animals have a significantly reduced expression of picd-1 than the wild-type animals, it is conceivable that further reduction in picd-1 cannot exacerbate the short-lived phenotype. Alternatively, it is plausible that picd-1 is not involved in lifespan maintenance. To explore this further, we examined the lifespan of picd-1 mutant and RNAi-treated animals in the absence of other mutations. Both gk3701 and bh40 alleles reduced the lifespan of the animals. picd-1(bh40) worms had a significantly reduced lifespan at both 20 °C and 25 °C, and picd-1(gk3701) exhibited a similar phenotype at 25 °C (Fig. 9C,D, Table 2). These results were also supported by the RNAi experiments. The analysis of age-associated biomarkers revealed a progressive age-associated decline in both body bending and Data represent a cumulative of two replicates (total n = 10 for each genotype shown in panels (A) and (B); and n = 500 for N2, 120 for pry-1(mu38) and 14 for pry-1(mu38); picd-1(bh40) double mutants shown in panel (C) and error bars represent the standard deviation. (A) Statistical analyses were done using two-way ANOVA with Tukey's multiple comparison test and recorded in Table S2. (B,C) Statistical analyses were done using oneway ANOVA with Dunnett's multiple comparison test and significant differences are indicated by stars (*): * (p < 0.05), ** (p < 0.01), *** (p < 0.001), and **** (p < 0.0001). n.s., not significant. www.nature.com/scientificreports/ pharyngeal pumping rates (Fig. 9E,F). Overall, the data suggest that while picd-1 does not enhance the phenotype of pry-1 mutants, the gene plays an essential role in maintaining the usual lifespan of animals.
We have previously shown that in addition to affecting lifespan, pry-1 regulates lipid metabolism 8,11 . This result prompted us to analyze whether picd-1 affects lipid levels and the expression of genes involved in fatty acid synthesis. The analysis of ∆9 desaturases showed that while fat-5 and fat-7 were downregulated, fat-6 was unaffected (Fig. 9G). Among the three transcription factors regulating the expression of ∆9 desaturases, nhr-80 (NHR family) was downregulated, but sbp-1 (SREBP1 homolog) was upregulated (Fig. 9G) 23 . We also quantified lipids by Oil Red O staining but saw no change in picd-1 mutants (Fig. 9H), possibly due to functional redundancies within the fat 24,25 and nhr family of genes 23 . Hence, we conclude that picd-1 is necessary for the expression of a subset of lipid synthesis genes but is not crucial for regulating lipid levels.
As pry-1 is necessary for picd-1 expression, we examined its effect on the CRTC-1::RFP and found that the fluorescence was nuclear-localized and dod-24 and asp-12 were upregulated in pry-1 mutants (Fig. 10D-F). To further understand the mechanism of PRY-1-PICD-1-mediated CRTC-1 localization, we examined the involvement of the Wnt canonical pathway component BAR-1/β-catenin. The bar-1 RNAi did not affect CRTC-1::RFP nuclear localization in pry-1 mutants (Fig. 10G,H), suggesting that PRY-1 may function in a WNT-independent manner to regulate CRTC-1. This conclusion agrees with our previous findings that pry-1-mediated lifespan maintenance does not depend on bar-1 10 . Next, we examined the involvement of CRTC-1 in PRY-1-mediated lifespan and stress response and found that crtc-1 RNAi performed during the adult stage significantly rescued the short lifespan and stress sensitivity defects of pry-1 mutants (Fig. 10I,J and Table 2). Hence, we propose that PRY-1 inhibits CRTC-1-dependent transcriptional response to delay age-associated processes.
Given that calcineurin and CRTC-1 mediated lifespan regulation involves the C. elegans CREB transcription factor homolog 1 (CRH-1), we wanted to know whether PRY-1 and CRH-1 regulate a common set of target genes. Consistent with our hypothesis, we found a significant overlap in differentially expressed genes between pry-1 and crh-1 mutant transcriptomes (406 common genes, R.F 2.5, p < 5.108e−78) 8,27 . Furthermore, the number of overlapping sets of genes regulated in an opposite manner between pry-1 and crh-1 mutants were also significant ( Figure S3 and Table S3) and were enriched with GO biological processes such as response to stress (18), metabolic processes (18), and cellular processes (40) (FDR < 0.05). In conclusion, these data demonstrate a novel functional relationship between PRY-1 and CRTC-1 in C. elegans.

Discussion
In this study, we identified a new gene, picd-1, in C. elegans that interacts with pry-1 and regulates several larval and adult processes. picd-1 is predicted to encode a nuclear protein containing a conserved Cabin1 domain, which belongs to the HUCA complex in humans 12 . The HUCA complex is implicated in diverse chromatin regulatory events, where it preferentially deposits a histone variant H3.3. This leads to transcriptional activation by nucleosome destabilization or transcriptional repression through heterochromatinization 28 . cabin1 is expressed in all human tissues and localized to the nucleoplasm and cytoplasm 29,30 . Studies in other systems have also uncovered homologous proteins of CABIN1. For example, the yeast Saccharomyces cerevisiae contains Hir1p and Hir2p (both HIRA orthologs) and Hir3, Hpc2, and Asf1p, orthologs of CABIN1, UBN1, and ASF1a, respectively 28 .
Our study provides the first genetic evidence of a Cabin1 domain-containing protein regulating biological processes in C. elegans. Other complex components in worms include HIRA-1 (HIRA homolog), ASFL-1, and UNC-85 (both ASF1a homologs) [31][32][33] . However, it remains to be seen if any of these proteins interact with PRY-1. Mutations in picd-1 led to multiple defects such as Pvl, Egl, small brood size, developmental delay, stress sensitivity, and short lifespan. While there are differences in phenotypic severity between two picd-1 alleles, it is important to emphasize that the loss of picd-1 function enhanced various phenotypes of the pry-1 mutant. For Table 2. Lifespan analysis of animals. Each lifespan assay was carried out in two or more batches (see Methods). Statistical analyses were done by comparing mutants with N2 control and RNAi treated animals with empty vector control. Comparisons of pry-1(gk3681); picd-1(gk3701) with pry-1(gk3682) and picd-1(gk3701) with picd-1(bh40) showed no significant differences. N, number of animals examined, n.s., not significant. www.nature.com/scientificreports/ example, pry-1; picd-1 double mutant showed a Pvl phenotype and exhibited P11.p cell fate changes. In addition, picd-1 RNAi enhanced seam cell defects in the pry-1 mutants. Interestingly, mutations in picd-1 did not enhance vulval precursor cell induction or Muv phenotype in pry-1 mutants. One possible model to explain genetic interactions between pry-1 and picd-1 is that pry-1 functions via both picd-1 dependent and independent pathways, and picd-1 is regulated by multiple factors. We analyzed the role of picd-1 in pry-1-mediated non-developmental events, such as egg-laying, embryonic survivability, aging, stress response, and lipid metabolism. Loss of picd-1 function worsened the embryonic lethality of pry-1 mutants. Moreover, pry-1; picd-1 double mutant had a very small brood size due to defects in the gonad arms. These findings suggest that pry-1 and picd-1 interact to regulate embryonic viability and fertility in animals. Similar phenotypes are observed in the mutants of other HIRA complex components. Knockdown of hira-1 leads to embryonic lethality, asfl-1 or unc-85 single mutants have low brood size, and asfl-1; unc-85 double mutant is sterile [32][33][34] . Together, these data show that pry-1 and picd-1 interact to regulate embryonic viability and fertility in animals. It remains to be seen whether PRY-1 and PICD-1 interact with other HIRA complex components to mediate their function.
Furthermore, we found that picd-1 is required for normal stress response maintenance. picd-1 mutants showed enhanced sensitivity to paraquat and tunicamycin. The mutant animals also exhibited increased levels of UPR markers. Both picd-1 and pry-1 mutants significantly increased hsp-16.2, and hsp-4, suggesting that these genes function together to regulate ER-UPR and HSR. However, more work is needed to determine whether these two genes uniquely affect MT-UPR and oxidative stress and their biological significance.
Mutants that show sensitivity to stress typically have a short lifespan [35][36][37] . Similar to pry-1 mutants, picd-1(bh40) animals are short-lived and exhibit defects in age-related physiological markers. This result is consistent with the fact that both genes function together to regulate stress response and aging. However, there are functional differences between the two genes. For example, we found that lipid levels were greatly reduced in pry-1 but not in picd-1 mutants. The nhr-80 and fat-7 levels were reduced in picd-1 mutant animals, consistent with the known role of nhr-80 in regulating fat-7 expression 24 . However, while picd-1 is needed for the expression of fat-5, fat-7 and nhr-80, a lack of its function does not compromise lipid levels in animals. These results are consistent with the above model of picd-1 participating in a subset of pry-1-mediated processes. However, the extent to which the two genes interact and the precise nature of their interactions is unknown.
A possible mechanism of picd-1 function in lifespan maintenance may involve calcineurin. AMPK and calcineurin modulation of CRTCs are conserved in mammals and C. elegans 17,[38][39][40][41] . In C. elegans, AAK-2 and calcineurin regulate CRTC-1 post-translationally in an opposing manner, where activated AAK-2 causes nuclear exclusion of CRTC-1 and extends lifespan. Such a phenotype was also observed after deactivating calcineurin 17 . Our data showed that loss of picd-1 function resulted in the nuclear localization of CRTC-1 and activated the CRTC-1 target genes. These findings, together with the fact that mammalian cabin1 inhibits calcineurin-mediated signaling 13,26,42 , allows us to hypothesize that PICD-1 regulates CRTC-1 via downregulation of calcineurin in C. elegans. According to this, loss of picd-1/cabin-1 is expected to increase calcineurin signaling, which may explain the shorter lifespan of picd-1 mutants. Further experiments are needed to investigate the extent to which the regulatory relationship between cabin1 and calcineurin is conserved in C. elegans.
As picd-1 is downregulated in pry-1 mutants, and both genes are needed for the proper subcellular localization of CRTC-1 and its downstream targets, we speculate that PRY-1 and PICD-1 use CRTC-1 to regulate stress response and lifespan of animals. While there is no evidence for the interaction between mammalian Axin and CRTCs, studies have shown that CREB, which associates with CRTCs, is inhibited by Axin-GSK3β signaling 43,44 . Our work provides the first evidence of genetic interactions between pry-1, picd-1, and crtc-1 in C. elegans, which has uncovered a novel crosstalk between Axin and calcineurin signaling. However, several questions remain unanswered. For instance, the components of pry-1 signaling affecting CRTC-1 nuclear localization are unknown. In preliminary experiments, tax-6 RNAi (calcineurin catalytic subunit) did not affect the pry-1 phenotype; however, more experiments are needed to completely ascertain its requirements. Additionally, whether picd-1 is regulated by pry-1 in a WNT-dependent manner or it is co-regulated by pry-1 and aak-2 4,10 independently of WNT needs thorough investigation. Moreover, it is unknown whether other HUCA complex components interact with PICD-1 to mediate PRY-1's role during stress response and lifespan, as well as whether PRY-1 and PICD-1 co-regulate a common set of targets during these processes. Further work is needed to investigate these questions and to gain a deeper understanding of the conserved mechanisms involved in Axin-CABIN1 signaling in eukaryotes. Mutant alleles and transgenic strains. The gk3701 mutation was discovered in a CRISPR screen that was carried out in Moerman lab to obtain alleles of pry-1 and several other genes of interest 6,45,46 . Since the original picd-1 mutant strain also contained pry-1(gk3681), it was deconstructed to establish separate strains of picd-1(gk3701) (outcrossed 3x) and pry-1(gk3681) (outcrossed 2x). The presence of CRISPR mutations was confirmed by PCR and sequencing. Molecular changes in pry-1(gk3682) were described earlier by our group 6 . Both gk3682 and gk3681 carry a CRISPR GFP cassette 45 We sequenced gk3681 as part of this study and found that the allele is identical to gk3682 (Table S4). In agreement with this, pry-1(gk3681) animals are phenotypically similar to pry-1(gk3682) ( Figure S1 and Table 2). The picd-1(gk3701) does not contain a CRISPR GFP cassette. Instead, it carries a 5 bp sequence (GGTGA) deletion in the second exon of the gene (flanking 25 nucleotides: GTG AAG AGG ATG AGG ACA ATG GTG A and GGA TTC AGA AGA AGA AGA TGA AGA A) that causes multiple premature in-frame stop codons.

Materials and methods
The other picd-1 allele (bh40) was generated in a separate CRISPR screen using a published protocol 47 . Here, we replaced the 84 bp in the first exon by a synthetic sequence containing stop codons in different reading frames. See primers in Table S4. bh40 animals were outcrossed 2 × and examined for various phenotypes. Another 2 × outcross of the strain was subsequently carried out (total 4x), which showed similar defects (Table S5).
To generate the picd-1p::GFP transgenic animals (DY678), pGLC150 was injected in N2 background along with myo-3::wCherry or dat-1::YFP marker. pGLC150 was constructed by cloning a 3,885 bp PCR-amplified fragment (using the primers GL1372 and GL1373), spanning the promoter region and a portion of the first exon of the picd-1 gene, into the vector pPD95.81 using the restriction sites SalI and KpnI. Molecular biology. RNA was extracted from synchronized L3 and day-1 adult animals. Protocols for RNA extraction, cDNA synthesis and qPCR were described earlier 7 . Briefly, total RNA was extracted using Trizol (Thermo Fisher, USA). The RNA was used to prepare cDNA and, subsequently, perform qPCR using the Sensi-Fast cDNA synthesis kit (Bioline, USA), and SYBR green mix (Bio-Rad, Canada), respectively. Primers are listed in Table S4.
RNAi. RNAi mediated gene silencing was performed using a protocol previously published by our laboratory 48 . Plates were seeded with E. coli HT115 expressing either dsRNA specific to candidate genes or empty vector (L4440). Gravid hermaphrodites were treated with a mild bleach solution (3:2 ratio of commercial bleach and 4 N NaOH) and eggs were plated. Animals were subjected to RNAi treatment from egg stage unless otherwise stated. Phenotypes were examined at the young adult stage. Fluorescent and DIC microscopy. Animals were paralyzed in 10 mM Sodium Azide and mounted on glass slides containing 2% agar pads and covered with glass coverslips. Images were captured using a Zeiss Apotome microscope and Zeiss ZEN software. For acquiring live videos of gonad, animals were suspended in M9 without Sodium Azide. Videos were captured by a high-speed camera fitted on a Leica MZ-FLIII fluorescent stereomicroscope 49 .
Seam cells were identified using scm::GFP and ajm-1::GFP markers, The nuclei of these cells were counted, and adult lateral alae were scored using Nomarski differential interference contrast and epifluorescence optics. Images were acquired using a Hamamatsu Camera mounted on a Nikon Eclipse 80i upright Nomarski fluorescence microscope and NIS Element software (Nikon, USA).

Vulval induction and P cells.
Vulval induction and P11.p and P12.p phenotypes were examined in mounted L4 stage animals using a Nomarski microscope. VPCs were considered induced if they gave rise to progeny. Wild-type (N2) animals have three induced VPCs, one each for P5.p, P6.p, and P7.p. Mutants with more than three induced VPCs were termed as 'over-induced' . Muv and Pvl phenotypes were scored in adults.  Table 2). (J) Bar graphs represent percentage survival of animals following 100 mM paraquat exposure for 2 h. Data represent a cumulative of two replicates (n > 60 animals) and error bars represent the standard deviation. For panels (B,E,G,J), statistical analyses were done using an unpaired t-test. Data for panels (C) and (F) were analyzed using Bio-Rad software (t-test). In all cases, significant differences are indicated by stars (*): * (p < 0.05), ** (p < 0.01), and **** (p < 0.0001). n.s., not significant. www.nature.com/scientificreports/ Vulval morphogenesis is described previously 50 . At the L4 stage VPC progeny undergo a series of changes that involve invagination of vulva progeny and selective cell fusion to give rise to a characteristic morphology of the vulva. Defects in morphogenetic processes can lead to an abnormal shape of the vulva. P11.p and P12.pa cells can be readily distinguished under a Nomarski microscope based on their nuclear size and morphology [51][52][53] . In pry-1 mutants, two P12.pa-like cells are observed and P11.p is missing.

Scientific
Aging-related analysis. Lifespan experiments of RNAi-treated animals were carried out using a previously described protocol 10 . Synchronized eggs were allowed to grow on NGM OP50 seeded plates till the late L4 larval stage after which they were transferred to RNAi bacteria seeded plates to perform adult stage-specific knockdown. Cultures were screened daily for dead animals and surviving adults were transferred every other day till the progeny production ceased. Censoring was done for animals that had either escaped, burrowed into the medium, showed a bursting at the vulva, or had progeny hatching inside the uterus 54 .
Body bending per 1 min and pharyngeal pumping per 30 s were analyzed in young adults over a period of four days 7 . For this, individual hermaphrodites were placed on OP50 plates and examined under a dissecting microscope. Pharyngeal pumping was assessed by observing the number of pharyngeal contractions. For body bending, animals were stimulated by tapping once on the tail by a platinum wire. Each full sinusoidal motion was counted as one body bend. Only animals that moved actively within 1 min were included in the analysis.
Stress assay and electrotaxis. Oxidative and endoplasmic reticulum mediated stress experiments were performed using 200 mM paraquat (unless specified) (Thermo Fisher Scientific, USA) and 25 ng/µl tunicamycin (Sigma-Aldrich, Canada) respectively. Animals were incubated for 1 h, 2 h, 3 h and 4 h, following a previous published protocol 6 . The final working concentrations were made in M9. At least 30 animals of each strain were tested in replicates. Means and standard deviations were determined from experiments performed in duplicate. Animals were considered dead if they did not respond to a platinum wire touch and showed no thrashing or swimming movement in M9. Moreover, dead animals usually had an uncurled and straight body shape in comparison to the normal sinusoidal shape of worms.
The electrotaxis assay protocol has been described previously 55 . Briefly, synchronized worms were introduced into a microfluidic channel and subjected to an electric field of 3 V/cm. Locomotory data was extracted from recorded videos using custom MATLAB-based worm tracking software. Electrotaxis speed data was plotted using box plots.
Oil Red O staining. Neutral lipid staining was done on synchronized day-1 adults using Oil Red O dye (Thermo Fisher Scientific, USA) following a published protocol 11 . Quantifications were performed using ImageJ software as described earlier 56 . Statistical analyses. SigmaPlot 11 was used to plot lifespan data that provided detailed statistical analysis on survival curves. The curves were estimated using the Kaplan-Meier test and differences among groups were assessed using the log-rank test. qPCR data analysis was done using software (Maestro 3.1) that controls the Bio-Rad CFX qPCR machine. The software performs built-statistical analyses of t-test and one-way ANOVA. For all other assays, data from repeat experiments were pooled and analyzed together, and statistical analyses were done using GraphPad Prism 9. p values less than 0.05 were considered statistically significant. Data for all the Figure panels are reported in Table S5.

Data availability
All data generated or analysed during this study are included in this published article (and its Supplementary Information files). Additional details are available from the corresponding author on reasonable request. www.nature.com/scientificreports/